a relatively short DNA sequence and as a consequence, they are too unspecific to be used directly for DNA engineering applications in cellula.
Novel improvements in DNA assembly methods combined with the cost reduction and with the increase in accuracy of DNA synthesis processes have led to the possibility of assembling large DNA constructs in vitro. Synthetic genomes will have a key role in future DNA engineering platforms but they will not be discussed in this chapter and in this book, where we will focus on in cellula genome engineering approaches.
In this chapter, I will give a brief description of the advancements in the precise genome editing field starting from observations of single‐stranded oligonucleotides‐mediated repair in yeasts to Recombineering and CRISPR‐Cas9‐dependent editing. These technologies have all greatly expanded the tools and methods that are used to generate disease models and to develop assays for drug discovery (Figure 2.1).
2.2 Bacterial DNA Engineering (Recombineering)
Microbes and microbial‐derived systems have been extensively used for the development of novel DNA engineering tools and for the application of these tools to DNA cloning. Restriction enzymes, recombinase systems such as CRE/Lox, integrases such as ΦC31‐Int, and the Cas9‐CRISPR system have all microbial origin. Recombinases and integrases‐based systems have been extensively used to engineer the mammalian genomes but we will not discuss them in this book that is focusing on scarless genome engineering systems. This chapter will focus on the development of Recombineering for bacterial engineering and its use in genome engineering with particular focus on applications in drug discovery.
The inspiration for the Recombineering (recombination‐mediated DNA engineering) method came from studies in yeast where the possibility to introduce an exogenous DNA cassette in yeast genome was demonstrated in the years 1978–1979. Yeasts DNA recombination methods used homology arms to target a gene without the need of double‐strand breaks (DSBs) generation or the expression of any exogenous protein (Hinnen et al. 1978; Scherer and Davis 1979). It was also demonstrated in yeasts that single‐stranded oligonucleotides with short homology arms to a target sequence are able to promote genomic insertions/deletions/modifications (Bhargava et al. 1999) Unfortunately, this approach does not work efficiently in wild‐type bacteria or in higher eukaryotes. This has limited the utility of single‐strand oligonucleotides‐mediated DNA editing of bacterial and mammalian genomes before the advent of recombineering. Murphy´s and Stewart´s groups showed for the first time that a portable recombination system can be introduced in Escherichia coli to induce recombination in bacteria in a similar fashion to the yeast recombination system but with much higher efficiency (Murphy 1998; Zhang et al. 1998). The portable cassette encodes for an exonuclease (i.e. Redα for the lambda Red system), a DNA annealing protein (i.e. Redβ), and the RecBCD inhibitor (i.e. Redγ). In particular, Stewart´s group showed that this system works with very short homology arms (as short as 30nt) via a peculiar mechanism of single‐strand heteroduplex intermediates at the replication fork (Maresca et al. 2010). This observation paved the way to the use of Recombineering for Precise Genome Editing of Bacterial Genome and for molecular cloning strategies. Recombineering overcomes the limitation of classical restriction/ligation‐based cloning because it does not require the availability of unique restriction sites in the target plasmid and it is specific enough to target the bacterial genome. Therefore, Recombineering has been extensively used for the seamless engineering of large constructs such as bacterial artificial chromosomes (BACs) and for the engineering of bacterial genome.
Figure 2.1 Graphical overview of genome engineering technologies (upper panel) and methods (lower panel) developed during the latest 27 years. A schematic representation of the different technologies or methods is presented, respectively, above or under the timeline arrow.
2.3 BAC Recombineering
The use of recombineering has been particularly important for functional genomics programmes where BAC transgenes or Gene Targeting constructs have been engineered at large scale to generate animal models of disease or to develop libraries of gene tagging. The European Conditional Mouse Mutagenesis (EUCOMM) and Knock‐Out Mouse Programme (KOMP) contributed to the large‐scale generation of conditional gene KO mice that have been extensively used in Drug Discovery. In particular, Skarnes and colleagues developed a large conditional knock‐out mouse library in the framework of the EUCOMM programme by using a high‐throughput gene‐targeting pipeline based on Recombineering (Skarnes et al. 2011). This gene‐targeting pipeline has been greatly facilitated by the development of a high‐throughput strategy of DNA engineering where “recombineered” targeting constructs were used to engineer C57BL/6N mouse embryonic stem cell for the generation of KO mice. This mouse library has been instrumental to understand the function of genes encoded by the mammalian genomes (In vivo) and to validate drug targets.
Another particular relevant example of Recombineering applications in Drug Discovery/Development is the remarkable work by scientists at Regeneron Pharmaceuticals aimed to engineer a humanized mouse model producing human–mouse hybrid antibodies. Their VelociGene platform (Murphy 1998) allowed the generation of multiple knock‐out/knock‐in by an high‐throughput recombineering & gene targeting approach where “recombineered” BACs are inserted in mESC using sequential homologous recombination steps. This led to the replacement of mouse immune genes with human orthologs (Valenzuela et al. 2003).
Finally, a very relevant application of Recombineering is the generation of tagged genes libraries. Gene tagging can potentially overcome the use of high‐affinity antibodies to detect gene expression, but it is limited by the lack of faithful gene activity of tagged protein generated with the use of overexpressed cDNA vectors. BAC transgenes guarantee a quas‐physiological level of gene expression maintaining transgene regulatory element and promoters, although the tagged gene is not integrated in its endogenous locus but in a so‐called third allele. The generation of tagged BAC libraries at scale was greatly simplified by selecting recombineering events in liquid bacterial culture. The potential of this system is exemplified by the generation of genome‐wide BAC libraries for the analysis of protein localization in Daino Rerio and Caenorhabditis elegans (Sarov et al. 2006).
2.4 Metabolic Engineering
The highly diverse chemical structures of Natural Products isolated from microbes or derived semisynthetically from natural intermediates allowed the development of a broad range of different drug activities, including antibiotics and chemotherapeutics.
Genome sequencing data facilitated by the development of Next Generation sequencing platforms indicate that microbial genomes contain an untapped resource of biosynthetic gene cluster that can be exploited to generate novel functions.
Unfortunately, most of these gene clusters are not expressed under normal laboratory growth conditions even when it is possible to grow the natural host in lab environment. In addition, the size of the Biosynthetic gene clusters (reaching up to 200kb) renders the in vitro manipulation of this large clusters difficult (Smanski et al. 2016).
Recombineering or Recombineering‐derived strategies have therefore been an ideal method to characterize and to engineer long gene clusters. In fact, specific gene clusters can be inserted in an heterologous host to facilitate the genetic manipulation of the genes present in these clusters.
An alternative strategy would be the use of endogenous recombineering systems from different hosts
